DNA hypomethylation silences anti-tumor immune genes in early prostate cancer and CTCs.

Cancer is characterized by hypomethylation-associated silencing of large chromatin domains, whose contribution to tumorigenesis is uncertain. Through high-resolution genome-wide single-cell DNA methylation sequencing, we identify 40 core domains that are uniformly hypomethylated from the earliest detectable stages of prostate malignancy through metastatic circulating tumor cells (CTCs). Nested among these repressive domains are smaller loci with preserved methylation that escape silencing and are enriched for cell proliferation genes. Transcriptionally silenced genes within the core hypomethylated domains are enriched for immune-related genes; prominent among these is a single gene cluster harboring all five CD1 genes that present lipid antigens to NKT cells and four IFI16-related interferon-inducible genes implicated in innate immunity. The re-expression of CD1 or IFI16 murine orthologs in immuno-competent mice abrogates tumorigenesis, accompanied by the activation of anti-tumor immunity. Thus, early epigenetic changes may shape tumorigenesis, targeting co-located genes within defined chromosomal loci. Hypomethylation domains are detectable in blood specimens enriched for CTCs.

TGF-ß in the microenvironment induces a physiologically occurring immune-suppressive senescent state.

TGF-ß induces senescence in embryonic tissues. Whether TGF-ß in the hypoxic tumor microenvironment (TME) induces senescence in cancer and how the ensuing senescence-associated secretory phenotype (SASP) remodels the cellular TME to influence immune checkpoint inhibitor (ICI) responses are unknown. We show that TGF-ß induces a deeper senescent state under hypoxia than under normoxia; deep senescence correlates with the degree of E2F suppression and is marked by multinucleation, reduced reentry into proliferation, and a distinct 14-gene SASP. Suppressing TGF-ß signaling in tumors in an immunocompetent mouse lung cancer model abrogates endogenous senescent cells and suppresses the 14-gene SASP and immune infiltration. Untreated human lung cancers with a high 14-gene SASP display immunosuppressive immune infiltration. In a lung cancer clinical trial of ICIs, elevated 14-gene SASP is associated with increased senescence, TGF-ß and hypoxia signaling, and poor progression-free survival. Thus, TME-induced senescence may represent a naturally occurring state in cancer, contributing to an immune-suppressive phenotype associated with immune therapy resistance.

Single-cell transcriptomic profiling for inferring tumor origin and mechanisms of therapeutic resistance.

Head and Neck Squamous Cell Carcinoma (HNSCC) is an aggressive epithelial cancer with poor overall response rates to checkpoint inhibitor therapy (CPI) despite CPI being the recommended treatment for recurrent or metastatic HNSCC. Mechanisms of resistance to CPI in HNSCC are poorly understood. To identify drivers of response and resistance to CPI in a unique patient who was believed to have developed three separate HNSCCs, we performed single-cell RNA-seq (scRNA-seq) profiling of two responding lesions and one progressive lesion that developed during CPI. Our results not only suggest interferon-induced APOBEC3-mediated acquired resistance as a mechanism of CPI resistance in the progressing lesion but further, that the lesion in question was actually a metastasis as opposed to a new primary tumor, highlighting the immense power of scRNA-seq as a clinical tool for inferring tumor origin and mechanisms of therapeutic resistance.

Differential Kinase Activity Across Prostate Tumor Compartments Defines Sensitivity to Target Inhibition.

Cancer therapy often results in heterogeneous responses in different metastatic lesions in the same patient. Inter- and intratumor heterogeneity in signaling within various tumor compartments and its impact on therapy are not well characterized due to the limited sensitivity of single-cell proteomic approaches. To overcome this barrier, we applied single-cell mass cytometry with a customized 26-antibody panel to PTEN-deleted orthotopic prostate cancer xenograft models to measure the evolution of kinase activities in different tumor compartments during metastasis or drug treatment. Compared with primary tumors and circulating tumor cells (CTC), bone metastases, but not lung and liver metastases, exhibited elevated PI3K/mTOR signaling and overexpressed receptor tyrosine kinases (RTK) including c-MET protein. Suppression of c-MET impaired tumor growth in the bone. Intratumoral heterogeneity within tumor compartments also arose from highly proliferative EpCAM-high epithelial cells with increased PI3K and mTOR kinase activities coexisting with poorly proliferating EpCAM-low mesenchymal populations with reduced kinase activities; these findings were recapitulated in epithelial and mesenchymal CTC populations in patients with metastatic prostate and breast cancer. Increased kinase activity in EpCAM-high cells rendered them more sensitive to PI3K/mTOR inhibition, and drug-resistant EpCAM-low populations with reduced kinase activity emerged over time. Taken together, single-cell proteomics indicate that microenvironment- and cell state-dependent activation of kinase networks create heterogeneity and differential drug sensitivity among and within tumor populations across different sites, defining a new paradigm of drug responses to kinase inhibitors. SIGNIFICANCE: Single-cell mass cytometry analyses provide insights into the differences in kinase activities across tumor compartments and cell states, which contribute to heterogeneous responses to targeted therapies.

APOBEC Mutagenesis Is Concordant between Tumor and Viral Genomes in HPV-Positive Head and Neck Squamous Cell Carcinoma.

APOBEC is a mutagenic source in human papillomavirus (HPV)-mediated malignancies, including HPV+ oropharyngeal squamous cell carcinoma (HPV + OPSCC), and in HPV genomes. It is unknown why APOBEC mutations predominate in HPV + OPSCC, or if the APOBEC-induced mutations observed in both human cancers and HPV genomes are directly linked. We performed sequencing of host somatic exomes, transcriptomes, and HPV16 genomes from 79 HPV + OPSCC samples, quantifying APOBEC mutational burden and activity in both host and virus. APOBEC was the dominant mutational signature in somatic exomes. In viral genomes, there was a mean of five (range 0-29) mutations per genome. The mean of APOBEC mutations in viral genomes was one (range 0-5). Viral APOBEC mutations, compared to non-APOBEC mutations, were more likely to be low-variant allele fraction mutations, suggesting that APOBEC mutagenesis actively occurrs in viral genomes during infection. HPV16 APOBEC-induced mutation patterns in OPSCC were similar to those previously observed in cervical samples. Paired host and viral analyses revealed that APOBEC-enriched tumor samples had higher viral APOBEC mutation rates (p = 0.028), and APOBEC-associated RNA editing (p = 0.008), supporting the concept that APOBEC mutagenesis in host and viral genomes is directly linked and occurrs during infection. Using paired sequencing of host somatic exomes, transcriptomes, and viral genomes, we demonstrated for the first-time definitive evidence of concordance between tumor and viral APOBEC mutagenesis. This finding provides a missing link connecting APOBEC mutagenesis in host and virus and supports a common mechanism driving APOBEC dysregulation.

Regulatory variants in TCF7L2 are associated with thoracic aortic aneurysm.

Thoracic aortic aneurysm (TAA) is characterized by dilation of the aortic root or ascending/descending aorta. TAA is a heritable disease that can be potentially life threatening. While 10%-20% of TAA cases are caused by rare, pathogenic variants in single genes, the origin of the majority of TAA cases remains unknown. A previous study implicated common variants in FBN1 with TAA disease risk. Here, we report a genome-wide scan of 1,351 TAA-affected individuals and 18,295 control individuals from the Cardiovascular Health Improvement Project and Michigan Genomics Initiative at the University of Michigan. We identified a genome-wide significant association with TAA for variants within the third intron of TCF7L2 following replication with meta-analysis of four additional independent cohorts. Common variants in this locus are the strongest known genetic risk factor for type 2 diabetes. Although evidence indicates the presence of different causal variants for TAA and type 2 diabetes at this locus, we observed an opposite direction of effect. The genetic association for TAA colocalizes with an aortic eQTL of TCF7L2, suggesting a functional relationship. These analyses predict an association of higher expression of TCF7L2 with TAA disease risk. In vitro, we show that upregulation of TCF7L2 is associated with BCL2 repression promoting vascular smooth muscle cell apoptosis, a key driver of TAA disease.

Oropharyngeal Squamous Cell Carcinoma Morphology and Subtypes by Human Papillomavirus Type and by 16 Lineages and Sublineages.

Oropharyngeal squamous cell carcinoma (SCC) is increasing in incidence and, in Western countries, strongly associated with transcriptionally-active high-risk human papillomavirus (HPV). Within HPV-positive tumors, there is wide morphologic diversity with numerous histologic subtypes of SCC. There are also variable degrees of keratinization, anaplasia, stromal fibrosis, and maturing squamous differentiation. Unlike in the uterine cervix, where associations between HPV types and lineages/sublineages within types have been investigated with some clear correlations identified, little to no data exists for oropharyngeal SCC. In this study, for a large cohort of oropharyngeal SCC patients, we performed RTPCR for high-risk HPV. For the HPV positive patients, we sequenced the DNA of the entire HPV16 genome and determined lineages and sublineages, correlating HPV status, genotype, and HPV16 lineages/sublineages with SCC subtype and various histologic features. Of the 259 patients, 224 (86.5%) were high-risk HPV positive, of which 210/224 (93.8%) were HPV type 16 and 6/224 (2.7%) HPV type 33. Of the four HPV16 lineages, A was the most frequent (192/214 or 89.8%) and of the HPV16 A sublineages, A1 was the most frequent (112/210 or 53.3%). Patients with HPV negative tumors were more often keratinizing vs other types (23/35 or 65.7%) and thus more likely to have more maturing squamous differentiation and stromal desmoplasia. There was no significant correlation between HPV type (16 versus other), between HPV16 lineage (A versus others), or HPV16 A sublineages (A1 or A2 versus others) and morphologic type of SCC nor the various morphologic features of anaplasia/multinucleation, degree of keratinization, nor amount of stromal desmoplasia. In summary, in our cohort, there was no correlation between the type of HPV, the HPV 16 lineage or sublineage, and any of the histologic features or morphologic SCC subtypes.

A comparative study of protein-ssDNA interactions.

Single-stranded DNA-binding proteins (SSBs) play crucial roles in DNA replication, recombination and repair, and serve as key players in the maintenance of genomic stability. While a number of SSBs bind single-stranded DNA (ssDNA) non-specifically, the others recognize and bind specific ssDNA sequences. The mechanisms underlying this binding discrepancy, however, are largely unknown. Here, we present a comparative study of protein-ssDNA interactions by annotating specific and non-specific SSBs and comparing structural features such as DNA-binding propensities and secondary structure types of residues in SSB-ssDNA interactions, protein-ssDNA hydrogen bonding and π-π interactions between specific and non-specific SSBs. Our results suggest that protein side chain-DNA base hydrogen bonds are the major contributors to protein-ssDNA binding specificity, while π-π interactions may mainly contribute to binding affinity. We also found the enrichment of aspartate in the specific SSBs, a key feature in specific protein-double-stranded DNA (dsDNA) interactions as reported in our previous study. In addition, no significant differences between specific and non-specific groups with respect of conformational changes upon ssDNA binding were found, suggesting that the flexibility of SSBs plays a lesser role than that of dsDNA-binding proteins in conferring binding specificity.

Construction of habitat-specific training sets to achieve species-level assignment in 16S rRNA gene datasets.

BACKGROUND: The low cost of 16S rRNA gene sequencing facilitates population-scale molecular epidemiological studies. Existing computational algorithms can resolve 16S rRNA gene sequences into high-resolution amplicon sequence variants (ASVs), which represent consistent labels comparable across studies. Assigning these ASVs to species-level taxonomy strengthens the ecological and/or clinical relevance of 16S rRNA gene-based microbiota studies and further facilitates data comparison across studies. RESULTS: To achieve this, we developed a broadly applicable method for constructing high-resolution training sets based on the phylogenic relationships among microbes found in a habitat of interest. When used with the naïve Bayesian Ribosomal Database Project (RDP) Classifier, this training set achieved species/supraspecies-level taxonomic assignment of 16S rRNA gene-derived ASVs. The key steps for generating such a training set are (1) constructing an accurate and comprehensive phylogenetic-based, habitat-specific database; (2) compiling multiple 16S rRNA gene sequences to represent the natural sequence variability of each taxon in the database; (3) trimming the training set to match the sequenced regions, if necessary; and (4) placing species sharing closely related sequences into a training-set-specific supraspecies taxonomic level to preserve subgenus-level resolution. As proof of principle, we developed a V1-V3 region training set for the bacterial microbiota of the human aerodigestive tract using the full-length 16S rRNA gene reference sequences compiled in our expanded Human Oral Microbiome Database (eHOMD). We also overcame technical limitations to successfully use Illumina sequences for the 16S rRNA gene V1-V3 region, the most informative segment for classifying bacteria native to the human aerodigestive tract. Finally, we generated a full-length eHOMD 16S rRNA gene training set, which we used in conjunction with an independent PacBio single molecule, real-time (SMRT)-sequenced sinonasal dataset to validate the representation of species in our training set. This also established the effectiveness of a full-length training set for assigning taxonomy of long-read 16S rRNA gene datasets. CONCLUSION: Here, we present a systematic approach for constructing a phylogeny-based, high-resolution, habitat-specific training set that permits species/supraspecies-level taxonomic assignment to short- and long-read 16S rRNA gene-derived ASVs. This advancement enhances the ecological and/or clinical relevance of 16S rRNA gene-based microbiota studies. Video Abstract.

New insights into protein-DNA binding specificity from hydrogen bond based comparative study.

Knowledge of protein-DNA binding specificity has important implications in understanding DNA metabolism, transcriptional regulation and developing therapeutic drugs. Previous studies demonstrated hydrogen bonds between amino acid side chains and DNA bases play major roles in specific protein-DNA interactions. In this paper, we investigated the roles of individual DNA strands and protein secondary structure types in specific protein-DNA recognition based on side chain-base hydrogen bonds. By comparing the contribution of each DNA strand to the overall binding specificity between DNA-binding proteins with different degrees of binding specificity, we found that highly specific DNA-binding proteins show balanced hydrogen bonding with each of the two DNA strands while multi-specific DNA binding proteins are generally biased towards one strand. Protein-base pair hydrogen bonds, in which both bases of a base pair are involved in forming hydrogen bonds with amino acid side chains, are more prevalent in the highly specific protein-DNA complexes than those in the multi-specific group. Amino acids involved in side chain-base hydrogen bonds favor strand and coil secondary structure types in highly specific DNA-binding proteins while multi-specific DNA-binding proteins prefer helices.

Clinical Implications of Identifying Pathogenic Variants in Individuals With Thoracic Aortic Dissection.

BACKGROUND: Thoracic aortic dissection is an emergent life-threatening condition. Routine screening for genetic variants causing thoracic aortic dissection is not currently performed for patients or family members. METHODS: We performed whole exome sequencing of 240 patients with thoracic aortic dissection (n=235) or rupture (n=5) and 258 controls matched for age, sex, and ancestry. Blinded to case-control status, we annotated variants in 11 genes for pathogenicity. RESULTS: Twenty-four pathogenic variants in 6 genes (COL3A1, FBN1, LOX, PRKG1, SMAD3, and TGFBR2) were identified in 26 individuals, representing 10.8% of aortic cases and 0% of controls. Among dissection cases, we compared those with pathogenic variants to those without and found that pathogenic variant carriers had significantly earlier onset of dissection (41 versus 57 years), higher rates of root aneurysm (54% versus 30%), less hypertension (15% versus 57%), lower rates of smoking (19% versus 45%), and greater incidence of aortic disease in family members. Multivariable logistic regression showed that pathogenic variant carrier status was significantly associated with age <50 (odds ratio [OR], 5.5; 95% CI, 1.6-19.7), no history of hypertension (OR, 5.6; 95% CI, 1.4-22.3), and family history of aortic disease (mother: OR, 5.7; 95% CI, 1.4-22.3, siblings: OR, 5.1; 95% CI, 1.1-23.9, children: OR, 6.0; 95% CI, 1.4-26.7). CONCLUSIONS: Clinical genetic testing of known hereditary thoracic aortic dissection genes should be considered in patients with a thoracic aortic dissection, followed by cascade screening of family members, especially in patients with age-of-onset <50 years, family history of thoracic aortic disease, and no history of hypertension.

Efficiently controlling for case-control imbalance and sample relatedness in large-scale genetic association studies.

In genome-wide association studies (GWAS) for thousands of phenotypes in large biobanks, most binary traits have substantially fewer cases than controls. Both of the widely used approaches, the linear mixed model and the recently proposed logistic mixed model, perform poorly; they produce large type I error rates when used to analyze unbalanced case-control phenotypes. Here we propose a scalable and accurate generalized mixed model association test that uses the saddlepoint approximation to calibrate the distribution of score test statistics. This method, SAIGE (Scalable and Accurate Implementation of GEneralized mixed model), provides accurate P values even when case-control ratios are extremely unbalanced. SAIGE uses state-of-art optimization strategies to reduce computational costs; hence, it is applicable to GWAS for thousands of phenotypes by large biobanks. Through the analysis of UK Biobank data of 408,961 samples from white British participants with European ancestry for > 1,400 binary phenotypes, we show that SAIGE can efficiently analyze large sample data, controlling for unbalanced case-control ratios and sample relatedness.

Biobank-driven genomic discovery yields new insight into atrial fibrillation biology.

To identify genetic variation underlying atrial fibrillation, the most common cardiac arrhythmia, we performed a genome-wide association study of >1,000,000 people, including 60,620 atrial fibrillation cases and 970,216 controls. We identified 142 independent risk variants at 111 loci and prioritized 151 functional candidate genes likely to be involved in atrial fibrillation. Many of the identified risk variants fall near genes where more deleterious mutations have been reported to cause serious heart defects in humans (GATA4, MYH6, NKX2-5, PITX2, TBX5)1, or near genes important for striated muscle function and integrity (for example, CFL2, MYH7, PKP2, RBM20, SGCG, SSPN). Pathway and functional enrichment analyses also suggested that many of the putative atrial fibrillation genes act via cardiac structural remodeling, potentially in the form of an 'atrial cardiomyopathy'2, either during fetal heart development or as a response to stress in the adult heart.

Genome-wide analysis yields new loci associating with aortic valve stenosis.

Aortic valve stenosis (AS) is the most common valvular heart disease, and valve replacement is the only definitive treatment. Here we report a large genome-wide association (GWA) study of 2,457 Icelandic AS cases and 349,342 controls with a follow-up in up to 4,850 cases and 451,731 controls of European ancestry. We identify two new AS loci, on chromosome 1p21 near PALMD (rs7543130; odds ratio (OR) = 1.20, P = 1.2 × 10-22) and on chromosome 2q22 in TEX41 (rs1830321; OR = 1.15, P = 1.8 × 10-13). Rs7543130 also associates with bicuspid aortic valve (BAV) (OR = 1.28, P = 6.6 × 10-10) and aortic root diameter (P = 1.30 × 10-8), and rs1830321 associates with BAV (OR = 1.12, P = 5.3 × 10-3) and coronary artery disease (OR = 1.05, P = 9.3 × 10-5). The results implicate both cardiac developmental abnormalities and atherosclerosis-like processes in the pathogenesis of AS. We show that several pathways are shared by CAD and AS. Causal analysis suggests that the shared risk factors of Lp(a) and non-high-density lipoprotein cholesterol contribute substantially to the frequent co-occurence of these diseases.

Genome-wide Study of Atrial Fibrillation Identifies Seven Risk Loci and Highlights Biological Pathways and Regulatory Elements Involved in Cardiac Development.

Atrial fibrillation (AF) is a common cardiac arrhythmia and a major risk factor for stroke, heart failure, and premature death. The pathogenesis of AF remains poorly understood, which contributes to the current lack of highly effective treatments. To understand the genetic variation and biology underlying AF, we undertook a genome-wide association study (GWAS) of 6,337 AF individuals and 61,607 AF-free individuals from Norway, including replication in an additional 30,679 AF individuals and 278,895 AF-free individuals. Through genotyping and dense imputation mapping from whole-genome sequencing, we tested almost nine million genetic variants across the genome and identified seven risk loci, including two novel loci. One novel locus (lead single-nucleotide variant [SNV] rs12614435; p = 6.76 × 10-18) comprised intronic and several highly correlated missense variants situated in the I-, A-, and M-bands of titin, which is the largest protein in humans and responsible for the passive elasticity of heart and skeletal muscle. The other novel locus (lead SNV rs56202902; p = 1.54 × 10-11) covered a large, gene-dense chromosome 1 region that has previously been linked to cardiac conduction. Pathway and functional enrichment analyses suggested that many AF-associated genetic variants act through a mechanism of impaired muscle cell differentiation and tissue formation during fetal heart development.

Improving power of association tests using multiple sets of imputed genotypes from distributed reference panels.

The accuracy of genotype imputation depends upon two factors: the sample size of the reference panel and the genetic similarity between the reference panel and the target samples. When multiple reference panels are not consented to combine together, it is unclear how to combine the imputation results to optimize the power of genetic association studies. We compared the accuracy of 9,265 Norwegian genomes imputed from three reference panels-1000 Genomes phase 3 (1000G), Haplotype Reference Consortium (HRC), and a reference panel containing 2,201 Norwegian participants from the population-based Nord Trøndelag Health Study (HUNT) from low-pass genome sequencing. We observed that the population-matched reference panel allowed for imputation of more population-specific variants with lower frequency (minor allele frequency (MAF) between 0.05% and 0.5%). The overall imputation accuracy from the population-specific panel was substantially higher than 1000G and was comparable with HRC, despite HRC being 15-fold larger. These results recapitulate the value of population-specific reference panels for genotype imputation. We also evaluated different strategies to utilize multiple sets of imputed genotypes to increase the power of association studies. We observed that testing association for all variants imputed from any panel results in higher power to detect association than the alternative strategy of including only one version of each genetic variant, selected for having the highest imputation quality metric. This was particularly true for lower frequency variants (MAF < 1%), even after adjusting for the additional multiple testing burden.

Effects of short indels on protein structure and function in human genomes.

Insertions and deletions (indels) represent the second most common type of genetic variations in human genomes. Indels can be deleterious and contribute to disease susceptibility as recent genome sequencing projects revealed a large number of indels in various cancer types. In this study, we investigated the possible effects of small coding indels on protein structure and function, and the baseline characteristics of indels in 2504 individuals of 26 populations from the 1000 Genomes Project. We found that each population has a distinct pattern in genes with small indels. Frameshift (FS) indels are enriched in olfactory receptor activity while non-frameshift (NFS) indels are enriched in transcription-related proteins. Structural analysis of NFS indels revealed that they predominantly adopt coil or disordered conformations, especially in proteins with transcription-related NFS indels. These results suggest that the annotated coding indels from the 1000 Genomes Project, while contributing to genetic variations and phenotypic diversity, generally do not affect the core protein structures and have no deleterious effect on essential biological processes. In addition, we found that a number of reference genome annotations might need to be updated due to the high prevalence of annotated homozygous indels in the general population.

Protein-altering and regulatory genetic variants near GATA4 implicated in bicuspid aortic valve.

Bicuspid aortic valve (BAV) is a heritable congenital heart defect and an important risk factor for valvulopathy and aortopathy. Here we report a genome-wide association scan of 466 BAV cases and 4,660 age, sex and ethnicity-matched controls with replication in up to 1,326 cases and 8,103 controls. We identify association with a noncoding variant 151 kb from the gene encoding the cardiac-specific transcription factor, GATA4, and near-significance for p.Ser377Gly in GATA4. GATA4 was interrupted by CRISPR-Cas9 in induced pluripotent stem cells from healthy donors. The disruption of GATA4 significantly impaired the transition from endothelial cells into mesenchymal cells, a critical step in heart valve development.